Project Summary Cytoprotective and restorative treatments for acute ischemic stroke (AIS) is a major unmet medical need. The current standard of care for stroke patients is centered on recanalization efforts to restore cerebral perfusion through the administration of tissue plasminogen activator (tPA) or mechanical thrombectomy. However, despite a recent extension of recanalization treatment window, an estimated <10% of patients would be eligible for thrombolysis or thrombectomy, and a large proportion of victims are left with significant functional impairments even after reperfusion therapy. Furthermore, reperfusion therapy increases the incidence of hemorrhagic transformation which often worsens stroke outcome. When administered during the acute stroke phase, ArunA Bio?s neural stem cell-derived extracellular vesicle (NSC EV) product is effective in reducing lesion size, mitigating the systemic immune response, inhibiting hemorrhagic transformation, and promoting functional recovery. NSC EVs can be administered after the reperfusion therapy treatment window (>24 hours post-stroke) either alone or adjunctive to tPA and/or thrombectomy. Therefore, NSC EV have the potential to improve clinical AIS treatment paradigms by providing clinicians with an off-the-shelf, cytoprotective, and neurorestorative biologic to bolster recovery after recanalization efforts. The overall goal of this Phase II project is to support an Investigational New Drug (IND) application for NSC EV as a treatment for AIS. In order to advance the commercialization of NSC EV, the successful completion of Phase II studies will enable IND filing by conducting definitive pharmacology and toxicology studies in stroked rats. These goals will be achieved in the following three specific aims: 1) assess NSC EV tissue distribution and pharmacokinetics in a rat stroke model, 2) determine NSC EV dose response and associated toxicity in a rat stroke model, and 3) submit IND application for a NSC EV first-in-human study. In Aim 1, stroked rats will be treated with a high dose of NSC EV tagged with a radioactive tracer. Rats will undergo longitudinal PET/CT imaging and blood sampling to determine the kinetics of NSC EV tissue distribution and clearance. In Aim 2, stroked rats will be treated with either a one-dose or three-dose regimen of NSC EV at varying concentrations, and lesion size and sensorimotor function will be measured to determine dose response. Rats treated with the highest NSC EV dose will undergo clinical and histopathological analysis to determine any NSC EV-associated toxicity. In Aim 3, the key components of the IND submission including the pharmacology/toxicology studies outlined here as well as CMC and clinical protocols will finalized. The resulting IND application will be submitted to FDA, which is required for first-in-human studies and eventual commercialization.